The long-term goals of this research program are to elucidate the functions and underlying mechanisms of estrogen signaling in the biology of normal development of mammary glands and mammary tumorigenesis. In our preliminary studies, we identified and cloned a 36-kDa novel variant of Estrogen Receptor (ER)-a (ER-a36) that mediates membrane-initiated estrogen-and antiestrogen-signaling. It inhibits genomic estrogen signaling mediated by ER-a and-(3. ER-a36 is expressed in 60% breast cancer specimens examined, and even in 79% tumors characterized as ER-negative breast cancers. We also found that breast cancer susceptibility gene 1 (BRCA1) negatively regulates ER-a36 expression. We will pursue these preliminary studies further through the following experimental approaches. 1. We will study the functions of ER-cc36 in membrane-initiated estrogen- and antiestrogen-signaling observed in established ER-negative breast cancer cells and in mammary transformation resulted from BRCA1 dysfunction using in vitro and in vivo cell growth and transformation assays combined with the siRNA knockdown approach. 2. We will study the functions of ER-cc36 in malignant growth and development of antiestrogen resistance in ER-positive breast cancer using MCF7 cell variants that express different levels of ER-a36. We will also study the mechanisms by which ER-a36 functions in estrogen/antiestrogen signaling using the microarray approach and biochemistry assays. 3. We will study the mechanisms by which ER-a36 regulates ER-a expression and function using biochemistry, the gel shift, ChIP and luciferase assays. We will also study the molecular mechanisms by which ER-a36 expression is regulated by ER-a, the MARK pathway and BRCA1 through structure and function analysis of the ER-a36 promoter using DNA-protein binding assays and the luciferase assay. The significance of these studies resides in the fact that they will provide important and novel information on the molecular mechanisms by which estrogen signaling functions in normal and transformed cell growth. Such knowledge will greatly advance our progress in prevention and treatment of human breast cancer.